Maternal flutamide exposure induces spermatogenic dysfunction in male offspring: The mechanistic role of RARα-Mediated NLRP3 inflammasome activation

Maternal exposure to endocrine-disrupting compounds (EDCs) is linked to male reproductive decline, yet the mechanisms underlying developmental programming of long-term reproductive toxicity remain elusive. This study investigated how prenatal exposure to the anti-androgenic fungicide flutamide impairs spermatogenesis in offspring. Pregnant Sprague-Dawley rats were treated with flutamide (25 mg/kg/d) during late gestation (GD12-GD20). Testicular morphology and spermatogenic markers (Stra8, PCNA) were assessed in male offspring at PND30 and PND60. RNA-sequencing (RNA-seq) identified differentially expressed genes, while RARα antagonism (AR7) and Retinoic Acid (RA) rescue experiments were conducted in vivo and in testicular organ cultures to validate the mechanism. Prenatal flutamide exposure significantly reduced sperm count and disrupted seminiferous tubule architecture. RNA-seq revealed significant alterations in the RARα signaling pathway and oxidative stress-related genes. Mechanistically, flutamide suppressed RARα expression, which in turn triggered NLRP3 inflammasome activation. Inhibition of RARα by AR7 mimicked flutamide-induced spermatogenic defects and oxidative stress, whereas RA treatment significantly attenuated these toxic effects by restoring RARα/NLRP3 homeostasis. Our findings demonstrate that the RARα-NLRP3 axis is a critical mediator of flutamide-induced developmental reproductive toxicity, highlighting RARα as a potential therapeutic target for mitigating EDC-induced male infertility.